Over the past few years, we have identified and characterized a number of genes with novel roles in regulating centrosome size and centriole duplication. All such szy genes were identified in a screen for factors that genetically interact with the kinase ZYG-1, a conserved master regulator of centrosome duplication. Analysis of individual szy genes has led to the identification of several molecular pathways that control centriole duplication by controlling the expression levels of centriole assembly factors. Recently we have focused our attention on a novel pathway that regulates centriole duplication. This pathway is dependent on the function of the chromodomain helicase CHD-1, which is known to positively and negatively regulate transcription. In vertebrate cells, its known targets include genes encoding components of the centriole duplication pathway. Interestingly, CHD-1 co-precipitates with the ZYG-1 ortholog Plx4 in Xenopus egg extracts (Hatch et al. 2010 JCB 191: 721) suggesting another mode through which it might regulate centriole duplication. Consistent with the idea that a similar mechanism operates in worms, we have found that deletion of the C. elegans chd-1 homolog suppresses the centriole duplication defect of the hypomorphic zyg-1(it25) mutant. We have also used CRISPR-based gene editing to construct an independent chd-1 null allele and have found that it also suppresses zyg-1(it25). Curiously, mutation of the ATPase domain of the endogenous protein leads to suppression, while mutation of the DNA-binding domain does not. Recently we have found that loss of chd-1 affects expression of certain components of the centriole assembly pathway. Specifically, loss of CHD-1 is accompanied by an increase in the levels of SPD-2 and SAS-6 proteins. Using quantitative RT PCT, we have found that spd-2 and sas-6 mRNA levels appear unaffected. To identify relevant genes regulated by CHD-1, we have used RNA-Seq to quantitate differences in gene expression between wild-type and chd-1(null) mutant worms. Surprisingly, only a handful of genes (ten) are strongly affected by loss of CHD-1. However, we found that eleven known szy genes are moderately down-regulated in the absence of CHD-1, six of which are involved in regulated proteolysis. Our data therefore suggests that CHD-1 regulates centriole duplication by promoting the expression of numerous negative regulators. Despite its strong sequence conservation, loss of CHD-1 does not cause an obvious defect, as chd-1(null) worms appear morphologically normal and fail to show any embryonic lethality. Recently however, we have discovered a genetic interaction between chd-1 and another zyg-1 suppressor. The transcription factor DPL-1, encoded by the dpl-1/szy-10 gene) also negatively regulates centriole duplication; similar to loss of chd-1, loss of dpl-1 both suppresses a zyg-1 hypomorphic allele and results in elevated SAS-6 levels. We have found that depletion of DPL-1 by RNAi can enhance suppression of zyg-1 by the chd-1(null) allele suggesting that the two genes function in distinct pathways. Interestingly we find that combining the chd-1(null) allele and dpl-1(RNAi) produces a synthetic embryonic lethality. We are currently investigating the possibility that simultaneous disrupting the CHD-1 and DPL-1 regulatory pathways leads to defects in centriole duplication. In a related project, we are employing biochemical and biophysical approaches to characterize centriole duplication; specifically, we have been investigating how ZYG-1 regulates (and is regulated by) downstream components of the centriole duplication pathway. SAS-5 and SAS-6 are coiled-coil-domain-containing proteins that form the structural scaffold of the centriole and require ZYG-1 for their incorporation into centrioles. These proteins are known to form dimers and higher order oligomers that are important for their function. However, the potential role of ZYG-1 in regulating the oligomeric state and intermolecular interactions of these proteins has not been investigated. We have expressed full-length recombinant proteins in E. coli and purified them to near homogeneity. We have found that ZYG-1 and SAS-5 physically interact in vitro and that ZYG-1 is capable of phosphorylating SAS-5 in vitro. Further we have found that other centriole components (SAS-6 and SAS-4) can modulate ZYG-1 kinase activity. Using mass spectrometry, we find that ZYG-1 phosphorylates SAS-5 on a number of highly conserved serine and threonine residues. Of particular interest, four of these phosphorylated residues are in a region predicted to bind SAS-4, suggesting that ZYG-1 might regulate SAS-4-SAS-5 interactions. To address this, we used site directed mutagenesis to create a series of non-phosphorylatable and phosphomimetic versions of SAS-5 and tested their ability to interact with both SAS-4 and ZYG-1. Strikingly, we found that SAS-4 and ZYG-1 bind to the same region of SAS-5 and that phosphorylation biases binding toward one or the other partner. That is, those phosphorylation events that promote SAS-4 binding, inhibit ZYG-1 binding, and vice versa. This suggests that a central event in centriole assembly involves a hand off of SAS-5 from ZYG-1 to SAS-4. To confirm that this mechanism is important in vivo, we have used CRISPR-based gene editing to mutate the phosphorylated residues in the endogenous sas-5 gene. A number of the mutants exhibit the expected phenotypes (embryonic lethality and sterility) indicating that our in vitro results are physiologically relevant. We are continuing to characterize this mechanism with the goal of obtaining a better understanding of how these factors interact on a molecular level to build a nine-fold symmetric centriole.